1. Field of the Invention
This invention relates to reaction containers that can be physically and chemically defined to control the flux of molecules of different sizes and charge. The invention also relates to methods for constructing small volume reaction containers through etching or a combination of etching and deposition. The methods allow for the fabrication of multiple devices that possess features on multiple length scales, specifically small volume containers with controlled porosity on the nanoscale. The invention further relates to the use of the reaction containers in sensing and the production or conversion of biological materials.
2. Description of the Related Art
Biochemical reactions such as protein synthesis and enzymatic conversion are fundamental to the function of living systems and are vital tools in industry and research. As our understanding of these reactions, and the living cellular systems in which they are carried out, has grown, the importance of cellular organization and efficiencies achieved by operating at the cellular scale have become more apparent. This, combined with a desire to understand “what constitutes life”, has led to efforts to create synthetic cellular-scale containers, cell mimics, in which basic biochemical processes can be sustained.
With respect to cell mimics, U.S. Pat. No. 7,641,863 and corresponding U.S. Patent Application Publication No. 2004/0173506 describe nanoengineered structures for controlling material transport (e.g., molecular transport). In one example form, the structure for controlling transport of a material includes a membrane enclosure having at least one outer wall and at least a portion of one outer wall comprises a plurality of spaced apart fibers having a fiber width of 250 nanometers or less. A material is located within the membrane enclosure, and the material has a physical or chemical property such that the material is selectively restricted by the fibers from passing from the inside to the outside of the enclosure.
The structure includes means for controlling transport of the material both into and out of the membrane enclosure. In one version of the structure, chemical derivatization of the fibers may be undertaken to affect the diffusion limits or effect selective permeability or facilitated transport. For example, a coating can be applied to at least a portion of the fibers. In another embodiment, individually addressable carbon nanofibers can be integrated with the membrane to provide an electrical driving force for material transport. U.S. Pat. No. 7,641,863 is incorporated herein by reference.
Chemical reactions within the cell depend on the cellular membrane, as it (1) defines the spatial extent (volume) of the cell, and (2) regulates the transfer of reactants and products between the cellular reaction volume and its surroundings. Within the small volume of the cell, passive transport of molecules via diffusion is rapid and facilitates the efficient mixing of molecules within the cell. Furthermore, because the cell can contain only a limited, small number of molecules, small changes in the numbers of molecules can lead to drastic changes in concentration within the cell. Such changes can lead to significant alterations in cellular function and fate. Beyond its passive role as a “container,” the cellular membrane plays a dynamic role in affecting internal concentrations by regulating the exchange of materials between the internal volume of the cell and the external microenvironment, both in concert with and against electrical and chemical potentials.
A growing body of work has examined the efficacy of biomimetic systems for carrying out biological processes in micro/cellular-scale systems. Such work includes the solution synthesis of liposomes and vesicles, serial creation of surface bound, single and networked vesicles, and microfluidic generation of cellular-scale droplets formed in multiphase fluidics and inorganic microscale containers. In general, these approaches have all sought to capture the basic function of the cellular membrane, defining small volumes in which diffusive transport is efficient, and the transfer of materials between the internal reaction volume and the external environment is regulated.
The use of vesicle bioreactors for carrying out cell-free transcription and translation for the production of green fluorescent protein (GFP) has been examined, and it has been found that reactions could be sustained for up to four days with the incorporation of an α-hemolysin pore that made the membrane permeable to external reactants. (See, Noireaux et al., Proc. Natl. Acad. Sci. U.S.A., 2004, 101, 17669-17674.) In a similar work, the expression of enhanced GFP in liposomes using a specific and well-defined set of minimal enzymes was successfully carried out. (See, Murtas et al., Biochem. Biophys. Res. Commun., 2007, 363, 12-17). Despite the success of these solution based approaches, the isolation of individual vesicles for monitoring individual reactions (addressability) and potential difficulties involving long term stability and storage of such vesicles in extreme conditions of tonicity (osmolarity), pH and temperature remain a challenge for the integration of these vesicle reactors into a sensing and actuation platform.
A related work (Karlsson et al., J. Phys. Chem. B, 2005, 109, 1609-1617), utilized vesicles formed on surfaces, which could be connected by small nanoscale vesicle channels, to create small reaction volumes and networks. Because each volume could be formed using a different microinjection pipette, each could be filled with individual reagents and scaled according to the desired function. The technique was highly effective, and allowed tuning of mass transport from one volume to the other by controlling the interconnecting channel width and length, establishing a predictable concentration gradient (flux) between reaction containers. The approach demonstrates improved addressability over conventional liposome and vesicle based approaches, allowing the progression of a reaction within a single volume to be monitored over time. However, the serial nature by which the reaction volumes are created, coupled with their inability to be stored over long periods makes the creation and use of many identical reaction volumes difficult. Furthermore, like vesicles synthesized in solution, the approach is likely to suffer from poor stability under more extreme reaction conditions.
Advances in soft lithography have made the study of reactions in microscale droplets created within lab-on-a-chip (LOC) systems accessible. Significant contributions have been made in the development and study of droplet generating and mixing systems. The exquisite control of droplet size and mixing afforded by microfluidics has even facilitated studies of reaction kinetics in sub-cellular-scale droplets. A review of the topic of reactions in droplets, highlighting the techniques and describing various applications for microfluidic droplet generation and mixing can be found at Song, et al., Angew. Chem., Int. Ed., 2006, 45, 7336-7356. While droplet-based microfluidics provide exquisite control of single reaction volumes, the multiphase nature of these systems can make the exchange of materials between reaction containers difficult.
Therefore, there is a need for a cellular-scale reaction container that can be spatially addressed for monitoring and filling, while allowing both the storage and exchange of chemical information across the reactor membrane.